The central nervous system contains a specialized group of glial cells, known as astrocytes. Mature optic nerve and retinal astrocytes play a metabolically supportive role for retinal ganglion cells, as well as maintain the blood-retinal barrier. These cells can also transform into a destructive component of the nervous system, known as reactive astrocytes, which form permanent barriers to axonal regeneration. Developing optic nerve astrocytes (astrocyte precursors) play a number of roles that are unique to the eye. First, astrocyte precursors act as an attractant for ganglion cell axons as they leave the eye to grow towards other target cells in the rest of the central nervous system. Second, a subpopulation of the astrocyte precursor cells migrates into the eye where they beckon to endothelial cells from outside the eye to enter and act as the substrate upon which the endothelial cells form the primary retinal vasculature. The long term goal of my laboratory is to understand the mechanisms by which certain growth and patterning factors, such as bone morphogenetic proteins (BMPs) and sonic hedgehog (SHH), direct differentiation and gene expression in normal and reactive astrocytes in the optic cup and nerve. The goal of the current proposal is to determine the molecular mechanisms by which Pax2, a gene critical in astrocyte differentiation, may be regulated by BMP7 and SHH during development. This application will test the hypothesis that SHH and BMP7 cooperate in the regulation of Pax2 in primary retinal astrocytes by moderating the activity of the suppressor protein TLX. The studies proposed in this application involve a multi-disciplinary approach, including 1) in vitro and in vivo assays to determine interactions of BMP and SHH pathway members with other regulatory DNA-binding proteins such as co-immunoprecipitation, chromatin immunoprecipitation, and luciferase assays, 2) in vitro and in vivo manipulations of BMP pathways, 3) characterization of the phenotypic properties of optic nerve astrocytes in vitro and in vivo using immunohistochemistry, Western blots, in situ hybridization, quantitative polymerase chain reaction and image analysis. Information derived from these studies will further our understanding of the mechanisms necessary for the development of retinal and optic nerve astrocytes and may also increase our understanding of how reactive astrocytes develop following injury or disease to the retina. PUBLIC HEALTH RELEVANCE: Developing astrocytes in the optic stalk are critical for the path finding of retina ganglion cells as they leave the eye and the development of retinal vasculature. One transcription factor, Pax2, is necessary and sufficient for the differentiation of astrocytes. Our lab has proposed a novel mechanism whereby the intracellular signaling pathways of two factors found in the developing optic stalk, bone morphogenetic protein 7 and sonic hedgehog, cooperate in the modulation of repressor protein TLX at the Pax2 promoter. Funding of this application would result in 1) increased knowledge of the mechanisms involved in the development of retinal astrocytes and 2) will enhance our understanding of how these factors may work in the rest of the eye, and 3) augment our understanding of how reactive astrocytes may form in disease and following injury of the nervous system and perhaps provide therapeutic targets to treat reactive astrocytes.